Compact detection module for flow cytometers

ABSTRACT

In one embodiment, a flow cytometer is disclosed having a compact light detection module. The compact light detection module includes an image array with a transparent block, a plurality of micro-mirrors in a row coupled to a first side of the transparent block, and a plurality of filters in a row coupled to a second side of the transparent block opposite the first side. Each of the plurality of filters reflects light to one of the plurality of micro-mirrors and passes light of a differing wavelength range and each of the plurality of micro-mirrors reflects light to one of the plurality of filters, such that incident light into the image array zigzags back and forth between consecutive filters of the plurality of filters and consecutive micro-mirrors of the plurality of micro-mirrors. A radius of curvature of each of the plurality of micro-mirrors images the fiber aperture onto the odd filters and collimates the light beam on the even filters.

CROSS REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of U.S. Provisional PatentApplication No. 62/366,580 titled COMPACT DETECTION MODULE FOR FLOWCYTOMETERS filed on Jul. 25, 2016 by inventors Ming Yan et al.,incorporated herein by reference for all intents and purposes.

This patent application is further related to application Ser. No.15/498,397 titled COMPACT MULTI-COLOR FLOW CYTOMETER filed on Apr. 26,2017 by David Vrane et al. that describes a flow cytometer into whichthe compact detection module may be used and is incorporated herein byreference for all intents and purposes.

FIELD

The embodiments of the invention relate generally to detection modulesof flow cytometers.

BACKGROUND

A flow cytometer generally has a viewing orifice that is illuminated byone or more lasers. The laser light from the one or more lasers strikesvarious fluorochrome-labelled particles passing through the orifice. Thefluorochrome-labelled particles are typically various biological cellsin a sample labeled with different flourochromes (fluorescent dyes) thatcan be analyzed to obtain information about the sample that can begeneralized to the whole. One or more optical detectors in the flowcytometer are used to sense the fluorescence (fluorescent light) that isemitted from the fluorochrome-labelled particles passing through theorifice that are struck by the laser light from the one or more lasers.

One or more different optical filters can be arranged before the emittedfluorescence from the fluorochrome-labelled particles reaches eachdetector. The optical filters are arranged in the emission fluorescencepath such that each detector sees only a specific bandwidth of lightassociated with expected fluorescence of flourochromes. That is, thebandwidth of any given filter takes advantage of a peak in the emissionspectra of a specific fluorescent dye. In this way, for any givenparticle, the collective signal from the detectors indicates the type offlourochrome or fluorochromes attached to a particle. The signalsdetected by the detectors from the emitted fluorescence allows quick andcomprehensive cellular classification of the various particles in thesample.

However, emission spectrum can overlap between dyes. This limits thenumber of different fluorochromes that can be simultaneously detected ona given particle by a single laser and detector. Because emissionbandwidths typically range in wavelengths between 30 nanometers (nm) and60 nm, conventional flow cytometers can usually detect no more than forfour or five fluorochromes per laser line. Increasing the number oflasers provides an expedient but costly way to increase the number offluorochromes that can be simultaneously detected.

Further complicating detection of the emitted fluorescent light is thefact that many dyes used to stain particles are excited over a range oflaser wavelengths differing from and greater than the typical 30 nm to60 nm bandwidth range. This can lead to signal crosstalk betweendetectors for the different lasers.

Prior flow cytometry fluorescence detection systems limited thedivergence by increasing the collimating lens focal length. However,this resulted in a larger diameter light beam that limited the number ofdetectors, such as to six detectors, depending on the size of the finalimage required. In these prior flow cytometry systems, the final imagesize was constrained by optical aberrations in collimating a large sizeof optical imaging, such as 800 micro-meters or microns (um), and broadband light (e.g., 400 nm-800 nm in wavelength) into a set of detectorsthat has a size less than 3 millimeter (mm) in diameter.

In another flow cytometry system, the incident light is re-imaged usingspherical micro-mirrors for each detector in a detector chain of a setof detectors in a row. Re-imaging avoids the diverging collimated lightproblem of the above mentioned flow cytometry system. However, thenumber of detectors is limited by the aberration introduced byreflections from the spherical micro-mirrors. Since the image sizeincreases along the detector chain, to increase the number of detectorchannels down the row in the detector chain, large area detectors arerequired resulting in a large flow cytometer that is bulky andexpensive.

Resolution of spectrally overlapping emission spectra is also central toincreasing the number of detectable fluorochromes. A detector array canbe used to identify fluorochromes based on a collective emissionsignature across multiple wavelengths to increase the number ofdetectable fluorochromes. In essence, the entire fluorescence signal ischromatically dispersed into an array of detectors either by diffractiongrating or prisms. In this way, the entire emission spectra isdiscretized across the detectors. Spectral deconvolution (unmixing) canbe used to calculate the contribution of the known individualfluorochrome spectra to the total collected signal. However, thisapproach to increasing the number of detectable fluorochromes has twomajor limitations.

The continuous, linear characteristics of a dispersion-element/detectorarray does not allow for bandwidths to be adjusted to take advantage ofthe true nature of fluorochrome spectra. Accordingly, the recognition ofthe wider bandwidth favors longer wavelength fluorochromes whileoverlooking details of shorter wavelength fluorochromes with compressedspectra. Additionally, scattered light from other lasers, if present, isunavoidably collected by the array of detectors. This scattered lightcompromises the fluorescence signal that is to be detected by adetector.

Accordingly, there is a need for further improvement in a flow cytometerto increase the number of detectable fluorochromes and better analyzefluorochrome-labeled particles in a sample.

BRIEF SUMMARY

The embodiments of the invention are summarized by the claims thatfollow below.

BRIEF DESCRIPTIONS OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the United States Patent andTrademark Office upon request and payment of the necessary fee.

FIG. 1 is a block diagram of a detection module of a flow cytometersystem.

FIG. 2A is a schematic diagram of an image array with successive channelreimaging.

FIG. 2B is a schematic diagram of a if image array with alternatechannel reimaging.

FIG. 3 is a schematic diagram of a compact detection module including a16 channel if image array for a modular flow cytometer system.

FIG. 4A is a magnified view of fluorescent light exiting at differentpoints over the diameter of the optical fiber.

FIG. 4B is a magnified view of a detector to convert optical signalsinto electrical signals.

FIG. 5 is a magnified view of a portion of the if image array.

FIG. 6 is a diagram of a detection module in a modular flow cytometersystem with a 16 channel if image array and 16 detector channels.

FIGS. 7A-7C are differing diagrammatic views of a detection module in amodular flow cytometer system with a pair of 8 channel if image arraysand a pair of 8 detector channels.

FIG. 8 is a perspective view of a if image array with sphericalmicro-mirrors.

FIGS. 9A-9B are cross section views of the if image array of FIG. 8.

FIG. 10 is a perspective view of a if image array with concaverectangular micro-mirrors.

FIG. 11 is a top side view of a portion of the if image array of FIG.10.

FIG. 12 is a perspective view of a mounting block adjacent imaging blockand a low cost thin outline detector that is used in the detectormodules of FIG. 6 and FIGS. 7A-7C.

FIG. 13 is a top view of an optical plate assembly in a modular flowcytometry system.

DETAILED DESCRIPTION

In the following detailed description of the embodiments, numerousspecific details are set forth in order to provide a thoroughunderstanding. However, it will be obvious to one skilled in the artthat the embodiments can be practiced without these specific details. Inother instances well known methods, procedures, components, and circuitshave not been described in detail so as not to unnecessarily obscureaspects of the embodiments of the invention.

The embodiments of the invention include a method, apparatus and systemfor a flow cytometer with a compact highly multiplexed detection module.

A flow cytometer with a compact detection module for fluorescence isdisclosed with an increased numbers of detectors and a minimal imagesize at the detectors compared to prior flow cytometers. Each detectormodule is fed by at least one laser. Multiple lasers can be supported bymultiple detector arrays in a compact manner. The increased number ofdetectors is made possible by careful control of incremental aberrationsin the detector array as light is transmitted through the detectionchain. The compact size of the compact detection module is achieved by areduced distance between the micro-mirrors and filters and carefuldownward scaling, thereby minimizing image degradation down the row orchain of micro-mirrors and filters in the imaging array.

Prior limitations can be overcome by use of an optical system withmultiple individual detectors and an adjustable progression of filterbandwidths. The optical system enables a concentration of spectralcollocation points to best resolve spectra of both long and shortwavelength dyes used to mark the particles analyzed by the flowcytometer. Fluorescence from excited particles are imaged into amultimode fiber by an objective lens with a high numbered aperture (NA).The broadband fluorescence exiting the multimode fiber is collimated andthen coupled into (imaged onto) multiple detectors. Collimation of thebroadband fluorescent light exiting the multimode fiber is a challenge.

Referring now to FIG. 1, a functional block diagram of a detectionmodule 100 for flow cytometers is shown. An advanced flow cytometer caninclude a plurality of detection modules. The detection module 100 is awavelength de-multiplexing system. The detection module 100 successivelyreflects and reimages the fluorescent light output 101A from an opticalfiber 102 in an image array 106. The image array 106 is a mechanicalimage array including a plurality of reflective mirrors 112A-112E and acorresponding plurality of long-pass dichroic filters 110A-110Esuspended in air by mechanical mounts. Generally, a dichroic filter isan accurate color filter used to selectively pass light of a range ofwavelengths of color light while reflecting other wavelengths of colorlight. Alternatively, the plurality of long-pass dichroic filters110A-110E can be band pass filters.

The image array 106 is capable of reflectively reimaging a fiber spot oflight N times (where N is greater than 2) while maintaining the opticalquality of the fiber spot at the end of the image array. Reimaging isfunction of recreating the original image with some aberration, areimage, at a surface such as that of each of the dichroic filters110A-110E. The detection module 100 further includes a plurality ofdetector channels 113A-113E respectively comprising a plurality ofobjective lenses 116A-116E, and a plurality of detectors 118A-118E withlight being in communication with each. Optionally, the plurality ofdetector channels 113A-113E can further include a plurality of bandpassfilters 114A-114E respectively to be sure the various desired wavelengthranges are detected by the plurality of detectors.

In the image array 106, the light 101A that is incident onto the firstmirror 110A is reflected by the mirror into reflected light 103A. Thereflected light 103A from the first mirror 110A passes through air andis incident upon a first long-pass dichroic filter 112A. The light 103Afrom the first mirror 110A is split up by the long-pass dichroic filter112A into a continued light portion 101B and a pass-through ortransmitted light portion 105A. The continued light portion 101B passesthrough air and is incident upon the next mirror in the series, mirror110B. The transmitted light portion 105A is coupled into the firstbandpass filter 114A of the first detector channel 113A. The transmittedlight portion 105A is cleaned up by the bandpass filter 114A and thencoupled into the optical detector 118A by the objective lens 116A. Thisprocess repeats for each stage (detector channel) in the image array106.

The image array 106 includes five stages that reimages five times oneach long-pass dichroic filter. It is still desirable to include agreater number of detectors. However, after more than 5 reimages, beamdistortion through the reflective mirrors can accumulate to the pointwhere the image quality at the last dichroic filter becomes highlydeteriorated. To make the zigzag configuration between mirrors anddichroic filters in the image array 106 perform properly with a greaternumber of detectors, it is desirable to minimize image degradation alongthe light path.

Minimizing image degradation in a detection module can be achieved usingtwo mechanisms. If the bending power of each reflective mirror in theimage array is reduced (e.g., by a factor of two—one half the bendingpower), image degradation can be reduced. If the number of times thatthe light beam is reimaged in the image array is reduced (e.g., by afactor or two—one half the number of times) image degradation can befurther reduced.

Reducing the bending angle at the mirrors reduces aberrations of alltypes. Since aberrations increase non-linearly with bending angle, theimprovement gained by switching from “fast focus mirrors” to “slow focusmirrors” is significantly better than 2×, allowing retention of imagequality through many more reflections. The image quality incident on alast detector in a chain of detectors can be further improved byreducing the number of times a light beam is reimaged in the imagearray. Instead or re-imaging at each dichroic filter for each detectorchannel, the incident light can be reimaged on every other dichroicfilter and detector channel (e.g., odd detector channels).

Reference is now made to FIGS. 2A-2B to describe image arrays 106A-106Bproviding an improvement in image quality by using a transparent blockwith micro-mirrors having differing radius of curvature. Generally, theimage array consists of an array of micro-mirrors and an opposing arrayof bandpass and/or dichroic filters for each detection channel. Thethickness L of the transparent block between the serial chain or row ofmicro-mirrors M(n),M(n)′ on one side and the serial chain or row ofdichroic filters D(n) on the opposing side in each case is the same.However, the focal lengths of the micro-mirrors M(n) and M(n)′ differ inthe image arrays 106A-106B of FIGS. 2A-2B.

The focal length f of the micro-mirrors M(n)′ in FIG. 2B is L while thefocal length f of the micro-mirrors M(n) in FIG. 2A is one half L. Thelarger focal length of the micro-mirrors M(n)′ in the image array 106Breduces the bending angle and aberrations in the imaging along theserial chain of mirrors. Furthermore, the image array 106B is a if imagearray with the given the thickness L of the transparent block and thefocal length of the micro-mirrors M(n)′ such that reimaging occurs onodd dichroic filters, such as dichroic filters D(3), D(5), D(7), throughD(n).

In FIGS. 2A-2B, spot A(1) through spot A(n) are the spot sizes (area)respectively on the dichroic filters D(1) through D(N). Spot A(0) is thefiber aperture of the multi-mode fiber from which the fluorescent lightis input into each array 106A-106B. In FIGS. 2A and 2B, the fiberaperture can be considered to be infinitesimally small to illustrate theimage conjugation properties of the two designs.

FIG. 2A illustrates an image array 106A having a plurality ofmicro-mirrors M(1) through M(N) and a plurality of long pass dichroicfilters D(1) through D(N), where N is a whole number value greater thanone representing the number of detector channels. Through reflection bythe micro-mirror M(1), light focused at a spot A(1) at dichroic filterD(1) is re-imaged through reflection of micro-mirror M(1) to focus thelight at spot A(2) at dichroic filter D(2). This is repeated by each ofthe micro-mirrors M(2) through M(N) down the serial chain or row. Theimage array 106A is a 2f image array.

FIG. 2B illustrates a if design for an image array 106B with a pluralityof micro-mirrors M(1)′ through M(N)′ and a plurality of long passdichroic filters D(1) through D(N). The micro-mirrors M(1)′ throughM(N)′ have a different radius of curvature than the radius of curvatureof micro-mirrors M(1) through M(N). The if image array 106B provides animprovement in image quality at the detectors (e.g., detectors 118A-118Ein FIG. 1) over that of 2f image array 106A.

The chain of micro-mirrors M(1)′ through M(N)′ in the if image array106B are designed to relay an image down the chain through the propertyof telescope optics. For example, light focused at spot A(1) at thedichroic filter D(1) is imaged to spot A(3) at the dichroic filter D(3)through a telescope effect of micro-mirrors M(1)′ and M(2)′. The evenspot, Spot A(2), is an intermediate spot in the collimating space.

The plurality of long-pass dichroic filters D(1) through D(N) canalternatively be passband or bandpass optical filters or include both adichroic optical filter and a bandpass optical filter in combinationtogether to assure a limited range of wavelengths of light pass through.Dichroic optical filters use the principle of thin-film interference andcan also be referred to as an interference filter. For each channel, thebandpass or passband optical filter is tuned to pass a differentselected range of wavelength of light (the passband) to each detectorand reflect the rest of the light wavelengths back to a micro-mirror inthe micro mirror array.

To provide a compact detector module, the image array 106B is formed outof a solid transparent material such as further explained with referenceto FIGS. 8 and 10. The solid transparent material of the transparentimaging block used for the if image array can be clear glass or clearplastic, for example, with mirrors and dichroic filters formed into oron the transparent material.

The design of if image array 106B and the design of 2f image array 106Acan be compared, under the conditions of the same thickness of the solidtransparent material, the same pitch and the same incident angle. Thepath distance between adjacent micro-mirrors in the 2f image array 106Aand if image array 106B is similar. In this case, the path distance isthe physical distance, not the conventional path length, where therefraction index of the material is taken into account. However, thefocal length of micro-mirrors in the 2f image array 106A is shorter (onehalf) than the focal length of the micro-mirrors in the if image array106B. Stated differently, the actual focal length of the micro-mirrorsin the if image array 106B is twice that of the focal length of themicro-mirrors in the 2f image array 106A due to the different radius ofcurvatures in the micro-mirrors. A longer focal length reduces thebending power in each reflection, which minimizes the aberrationintroduced in the micro-mirror reflection. Accordingly, the aberrationsin the if image array 106B are improved over those of the 2f image array106A.

In the 2f image array system 106A, the fiber aperture A(0) is imaged tospot A(1) at the dichroic filter D(1). The image at spot A(2) is are-image of spot A(1) through reflection of micro-mirror M(1).Continuing through the zigzag light path in the array 106A, each spotA(n) at each dichroic filter D(n) is imaged by the next micro-mirrorM(n) to the next spot A(n+1) at the next dichroic filter D(n+1). In thisconfiguration, the path distance between spot A(n) and micro-mirror M(n)is the thickness L of the transparent block which is 2 times (twice) thefocal length of the micro-mirrors M(n).

In the if image array system 106B, light from the fiber aperture A(0) isimaged down to a spot A(1) at the dichroic filter D(1) by an inputchannel. In the image array system 106B, one can consider that the lightpath from micro-mirror M(1)′ to micro-mirror M(2)′ forms the equivalenceof a telescope with a magnification equal to 1. In such a case, the spotA(1) is imaged to spot A(3) through the telescope of micro-mirrors M(1)′and M(2)′ with spot A(2) being the intermediate spot in the collimatingspace. Adjacent micro-mirrors M(3)′ and M(4)′ form another telescope toreimage spot A(3) to spot A(5). Continuing the zigzag path in in theimaging array 106B, the odd spots A(1), A(3), A(5) . . . A(2n+1) are allconjugated to each other, with the even spots A(2), A(4), A(6), . . .A(2n) being intermediate spots in the collimating space. In thisconfiguration, the path distance between spot A(n) and micro-mirrorM(n)′ is the thickness L of the transparent block which is one focallength of the micro-mirrors M(n)′.

Each spot on the filter is formed by a bunch of light rays. The angulardistribution of the bunch of light rays is determined by the numericalaperture (NA) of the input multimode-fiber 102 and the input channel tothe imaging array of the imaging system. The cone-angle of light at spotA(1) is proportional to the numerical aperture of the multi-mode fiber102. If the image magnification m from the fiber aperture A(0) to thespot A(1) is a ratio of 1 to m, where m is greater than one, then thecone angle at spot A(1) is smaller than the cone angle at the fiberaperture A(0) by a factor of m.

In the 2f image array system 106A of FIG. 2A, the image magnificationfrom any spot A(n) to the adjacent spot A(n+1) is equal to one, for anyvalue of n. Disregarding the aberration, the cone angle of rays at thedichroic filters D(1) through D(N+1) of the image array system 106A arethe same for all detector channels.

Referring now to FIG. 2B, the distance between the dichroic filter D(1)and the micro-mirror M(1)′ is L. If the total number of channels N is aneven number in the if image array 106B, the spot A(1) is imaged to spotA(3) through micro-mirrors M(1)′-M(2)′ (considering a 1× telescope), andspot A(2) at dichroic filter D(2) is in the collimating space. A portionof the collimated light at spot A(2) is reflected by the dichroic filterD(2) towards the micro-mirror M(2)′.

Accordingly, the if image array 106B has odd detector channels at spotsA(1), A(3), . . . A(2k−1) . . . through A(N−1) and even detectorchannels at spots A(2), A(4), . . . A(2k), . . . through A(N), where1≦k≦(N/2). Since spot A(1) is in the front focal point of micro-mirrorM(1)′, and the path distance between adjacent micro-mirrors is twice thefocal length, all the odd spots A(1), A(3), A(5) . . . A(2n+1) are theimages of the fiber aperture (as indicated in FIG. 2B by light rayconvergence to the odd spots), and all the even spots A(2), A(4), A(6),. . . A(2n) are in the collimating space (as indicated in FIG. 2B by theparallel light rays at the even spots). Accordingly, as shown in FIG. 5,the cone angle of odd spots (the odd cone angle CAO) for odd detectorchannels is different from the cone angle of even spots (the even coneangle CAE) for the even detector channels.

In the if image array 106B, the center wavelength and passband width ofeach optical dichroic filter D(1) through D(N) differs from each other.The center wavelength and passband width of each optical dichroic filterare designed for optimized sampling of the fluorescence spectrum of dyeto yield better accurate unmixing of a large quantity of different dyes.For example, assuming a fluorescence spectrum of light wavelengths from400 nm to 800 nm and a sixteen (16) channel detection module, abandwidth of 25 nm in light wavelength can be analyzed by each. Forexample, the first detector channel and the first dichroic filter D(1)can bandpass and analyze light wavelengths from 400 nm to 425 nm with acenter wavelength of 412.5 nm. Wavelengths outside 400 nm to 425 nm aresubstantially filtered out and not passed onto the first detector in thefirst detector channel. A second detector channel and the seconddichroic filter D(2) can bandpass and analyze light wavelengths from 425nm to 450 nm with a center wavelength of 437.5 nm and increasing so onand so forth for each detector channel. The last or sixteenth detectorchannel and sixteenth dichroic filter D(16) can bandpass and analyzelight wavelengths from 775 nm to 800 nm with a center wavelength of787.5 nm.

The characteristics of the if image array 106B allow the initial opticalsignal to be propagated into a greater number of detectors than the 2fimage array 106A. The if image array 106B reduces the off-axisaberration through reducing the bending power in each mirror reflection.However, with the odd cone angles CAO being different from the even coneangles CAE at the dichroic filters in the odd and even channels, oneneeds to determine an optimum magnification m in an input stage from thefiber aperture A(0) to the spot A(1) at the dichroic filter D(1). For agiven fiber numeric aperture (NA) and aperture diameter, themagnification m from the fiber aperture A(0) to the spot A(1) at thedichroic filter D(1) is optimized for both odd and even detectorchannels in the if image array 106B.

From the spectrum resolution point of view, dichroic filter performancedegrades as the cone angle of the incident spot increases. In the ifimage array 106B, the cone angle of spots in odd channels are differentfrom that in the even channels. Basically, the cone angle in the oddchannels is determined by the numeric aperture (NA) of the multi-modefiber and the magnification factor m from fiber aperture A(0) to thespot A(1) at the dichroic filter D(1). In contrast, the cone angle ofeven channels is determined by the spot diameter of spots in the oddchannels.

In the input channel, assume the image magnification is m from apertureA(0) at the fiber to spot A(1) at the dichroic filter D(1). At the evendetector channel (dichroic filters D(2k)), the cone angle isproportional to the magnification m. However, in the odd detectorchannels (dichroic filters D(2k−1)), the cone angle is inverselyproportional to the magnification m. A larger magnification from thefiber aperture A(0) to the spot A(1) results in a smaller cone angle atodd detector channels (dichroic filters D(2k−1)) but a larger cone angleat the even detector channels (dichroic filters D(2k)). In one exampleembodiment having a multimode fiber with NA=0.12, aperture diameter 600um, and a micro-mirror (filter) pitch of 5.5 mm; the recommendedmagnification m was modeled to be approximately 2. Obviously, othermagnifications m can be determined with different inputs and thus theembodiments disclose herein are not limited to a 2× magnification. Inthe example presented for the if image array 106B, both the numericaperture (NA) and the number of reimages is reduced by a factor of 2,allowing for at least four times (4×) more detectors along a row of thesame length than the 2f image array 106A.

FIGS. 3, 4A-4B, and 5 illustrate graphs of simulation results of acompact detection module with the if image array 106B and example inputvalues of an embodiment having a fiber with NA=0.12, an aperturediameter 600 um, a micro-mirror (filter) pitch of 5.5 mm; and arecommended magnification of 2×. The different colors of light raysshown in FIGS. 3, 4A-4B, and 5 are for clarity only to show how light atthe different positions pass through the detector module.

FIG. 3 shows one end of the optical fiber 102 that launches fluorescentlight into the detection system. Near the opposite end (not shown) ofthe optical fiber 102, a collection objective lens with a high NA can beused to collect the fluorescent light from an aperture and couple itinto the opposite end of the optical fiber. The optical fiber 102 thencollects the light from the objective lens and directs it to the endshown in FIG. 3. Near the end shown in FIG. 3, the system can include afiber numeric aperture converter to lower the numeric aperture into freespace to launch the fluorescent light to the detector array.

Referring now to FIG. 3, the magnification m in the compact detectionmodule 300 is achieved by an input stage 301. The input stage 301includes a collimating lens 302, a blocking filter 303, and a focusinglens 304. The magnification, m, is achieved by adjusting the ratio offocal lengths of the collimating lens 302 and focusing lens 304. Forinstance, to set the magnification equal to 2 (m=2), the focal length offocusing lens 304 is twice that of the collimating lens 302. The inputchannel 301 may be considered to further include an input portion of atransparent block (e.g., wedge, block thickness, see FIG. 8) in theimage array 106B before the first dichroic filter D(1) is reached.

The collimating lens 302 receives the light launched from the fiber 102and collimates the light. The collimated light is passed through theblocking filter 303 and input into the focusing lens 304. The blockingfilter 303 is used to clean out the laser light that is scattered intothe collection optics near the opposite end of the optical fiber 102.The fluorescent spectrum of light associated with the fluorochromespasses through the blocking filter 303 and into the focusing lens 304.The focusing lens 304 focuses the fluorescent spectrum of light onto thefirst dichroic filter D(1) in the image array 106B to form an image atspot A(1). The image at spot A(1) is magnified in size (e.g., diameterand area) from the size at the aperture A(0) at the fiber by the factorm. The position of the lenses 302,304 between the end of the fiber 102and the image array 106B can be adjusted.

The compact detection module 300 further includes a sixteen (16) channelif image array 106B and sixteen (16) detector channels 313A-313P incommunication with the image array 106B (e.g., see FIG. 6). In analternate embodiment, a pair of eight (8) channel if image arrays (e.g.,see FIGS. 7A-7C) can be used in parallel to relax the imagingrequirements of each compact image array. In a flow cytometer, more thanone of these compact image arrays (e.g., three) can be used to multiplythe number of detector channels to be greater than sixteen (e.g., threetimes sixteen for forty detection channels) such as explained with thedetector modules described with reference to FIG. 13).

The image array 106B is formed out of a solid transparent blockmaterial. The sixteen (16) channel if image array 106B includes sixteen(16) dichroic filters D(1) through D(16) on one side of the transparentblock, and fifteen mirrors M(1) through M(15) on an opposite side. Theimage array does not need a mirror to follow after the last detectorchannel 313P. Moreover, the last filter D(16) 314 may not be a dichroicfilter; instead a bandpass filter can be used. In the case of thebandbass filter, the incident light need not be further reflected toanother mirror or filter.

Each detector channel 313A-313P (collectively referred to as detectorchannel 313) in the array or detectors includes a focusing lens 316, anda detector 318 (an instance is shown in FIG. 3). The detector 318 ispackaged in a thin outline (TO) can package 320 with the focusing lens316 coupled to or integrated into the TO can package. The focusing lens316 focuses the fluorescence light passing through the filter down ontoa small area size of the detector 318.

Referring now to FIG. 4A, the optical fiber 102 used to transportfluorescent light signals captured from the image chamber to thedetector array is a multi-mode optical fiber. Light exits the endsurface of a multimode fiber from various (if not all) locations over adiameter of the fiber, such as locations X1 through X5 for example. Thelenses 303,304 in the input channel shown in FIG. 3, focus light withinan aperture A(0) down to a spot A(1) on the first dichroic filter D(1).Because there is a two times (2×) magnification from spot A(0) to spotA(1), the spot size at the aperture A(0) is smaller than the spot sizeat spot A(1). The different colors for the light rays emanating from thedifferent locations X1 through X5 within the aperture shown in FIG. 4Aare for clarity only to show how light at the different positions passesthrough the detector module. As shown in FIG. 4A, an optical axis 402extends out from the circular center of the end of the optical fiber102. Light is launched out the end of the optical fiber 102 at a launchcone angle (CA) 404 with respect to the optical axis 402.

FIG. 3 shows the simulation results of the image array 106B and howlight from different locations in the optical beam are alternativelyimaged and collimated through the multiple reflections of the mirrorsand dichroic filters. While these results show all of the lightreflecting, the dichroic filters D(n) at any particular position differand allow transmission of a light signal (only shown in the lastdetector channel 313P in FIG. 3) according to their respective passband.

Referring now to FIGS. 3 and 4B, in each detector channel 313, thedesired wavelength range of the light signal passing through a dichroicfilter D(n) can be collected through a lens 316 and detected by a smallaperture photosensitive detector 318. A further bandpass filter 314 canalternatively or further be used in each detector channel. Otherwavelengths of light at the dichroic filter D(n), if any, are reflectedto the next micro-mirror M(n) along the chain or row of micro-mirrors.The row or chain of dichroic filters D(n) de-multiplexes differentranges of light wavelengths into the chain of detector channels313A-313P.

The magnified view of FIG. 5 shows simulation results of how the opticallight beam is alternatively imaged and collimated through the reflectionsurfaces of the micro-mirrors and dichroic filters. On the odd numbereddichroic filters (e.g., dichroic filters D(7), D(9), and D(11) shown inFIG. 5), the spots are the images of the fiber aperture. That is, thelight launched from the fiber aperture is imaged to each filter surfaceof the odd numbered dichroic filters. On the even numbered dichroicfilters (e.g., dichroic filters D(8), D(10), and D(12) shown in FIG. 5),the even numbered spots (e.g., spots A(8), A(10), and A(12) shown inFIG. 5) are in collimating spaces, where light rays emitting from apoint at the fiber aperture becomes a collimated beam. The beamdirections in the collimating space at each even numbered dichroicfilter are slightly different for different points from the fiberaperture.

In a flow cytometer, one or more linear 16-channel compact wavelengthdetection modules can be used to detect fluorescent signals of lightassociated with particles. Alternatively or conjunctively, one or moredual 8-channel compact wavelength detection modules can be used in aflow cytometer to detect fluorescent signals of light associated withparticles.

FIGS. 6 and 7A-7C illustrate embodiments of compact wavelength detectionmodules having the functionality of the if image array 106B shown inFIG. 2B. FIG. 6 illustrates a linear 16-channel compact wavelengthdetection module 600. FIGS. 7A-7C illustrate a dual 8-channel compactwavelength detection module 700.

Referring now to FIG. 6, linear 16-channel compact wavelength detectionmodule 600 includes an input stage (head) 601 and a detection module 614mounted to a base 610. Light is coupled into the input stage (head) 601by an optical fiber 102. The input stage (head) 601 includes acollimating lens 602, a long pass filter 603, a cleanup optical blocker604, and a focusing lens 605 mounted to an optical bench. The inputstage (head) 601 sets up the magnification m of the initial spot sizeimage A(1) on the first dichroic filter.

From the input stage 601, the light is coupled into a detection module614. An end of the input stage (head) 601 is coupled to a transparentwedge 607 to receive the light from the focusing lens 605. The inputstage (head) 601 and the detection module 614 are coupled to a chassisor base 610 of the flow cytometer to maintain their alignment together.

The detection module 614 includes a if image array 608 and adetector/lens array 611. The image array 608 is an embodiment of theimage array 106B of FIGS. 2B and 5. The image array 608 includes atransparent block 680 (e.g., see blocks 806,1006 of FIGS. 8 and 10)including the wedge 607 and fifteen micro-mirrors 612 on one side. On anopposing side of the transparent block 680, there are sixteen dichroicfilters 609. The detector/lens array 611, an embodiment of the pluralityof detectors 313A-313P, includes a plurality of photodetectors D1through D16 (e.g., detector 318 of FIG. 3) each having a lens (e.g.,lens 316 of FIG. 3) to focus the demultiplexed light into thephotodetector.

The light that is coupled into the image array 608 by the input stage601, is wavelength demultiplexed into the detectors D1 through D16 ofthe detector/lens array 611. The 16-channel detection module analyzes arange of wavelengths (e.g., 400 nm to 800 nm wavelengths).

To provide a different footprint that better fits a test bench andprovide parallel processing, the linear 16-channel compact wavelengthdetection module 600 can be instead implemented as a dual 8-channelcompact wavelength detection module.

Referring now to FIG. 7A, a top view of a dual detection module 700 isshown with a pair of 8-channel compact wavelength detection modules714,715. The compact wavelength detection module 700 includes an inputstage (head) 701 in communication with the first 8-channel detectionmodule 714 and the second 8-channel detection module 715 all of whichare mounted in alignment to a base 710. The first 8-channel detectionmodule 714 de-multiplexes and analyzes in parallel a first range ofwavelengths (e.g., 650 nm to 800nm—red wavelengths). The second8-channel detection module 715 de-multiplexes and analyzes in parallel asecond range of wavelengths (e.g., 400 nm to 650nm—blue wavelengths).

The light launched from the optical fiber 102 is coupled into the inputstage (head) 701. The light from the fiber 102 passes through acollimating lens 702 into a long pass dichroic filter 703. The long passdichroic filter 703 reflects light at the laser excitation wavelength(e.g., less than 400 nm) at a 45-degree angle to a scatter detector (notshown). The side scatter (SSC) light can be focused onto a smallaperture detector with a ball lens similar to that described for thefluorescent light. Fluorescent light in the fluorescent light spectrum(e.g., 400 nm-800 nm) passes through the long pass filter 703 and into asecond cleanup filter 704. The cleanup filter 704 ensures that noexcitation laser light reaches the de-multiplexing detection modules714-715.

After the cleanup filter 704, the fluorescent light is separated into along-wavelength band and a short-wavelength band by a long pass filter705. The long-wavelength light (e.g., red—650 nm to 800 nm) passesthrough the long-pass filter 705 and is focused into the first detectionmodule 714 by a collimating/focusing lens 706. The long wavelengthportion of light that passes that is focused by focusing lens 706 isde-multiplexed by the first detection module 714. The short-wavelengthlight band (e.g., blue—400 nm to 650 nm) is reflected by the long passfilter 705 back at an angle into a collimating/focusing lens 713. Thecollimating/focusing lens 713 focuses the short wavelength band of lightinto the second detection module 715. The short wavelength portionreflected by long-pass filter 705 is de-multiplexed by the seconddetection module 715. Alternatively, the filter 705 can be a short-passfilter and the short wavelength light passes through the filter and isde-multiplexed by the first detection module 714 while long wavelengthlight is reflected by the filter and is de-multiplexed by the seconddetection module 715.

Referring to the first detection module 714, light from the focusinglens 706 enters normal to a 12-degree wedge face 707, and passes throughthe transparent block (e.g., block 806 of FIG. 8) of the image array708, before imaging onto the first dichroic or bandpass filter 709.Light passes through the bandpass filter 709 and is focused onto a firstsmall area detector D1 in the detector/lens array 711. The lightrejected by the bandpass filter 709 is reflected back onto a firstmicro-mirror M(1) 712 of a plurality of micro-mirrors M(1) through M(7)in the image array. The first micro-mirror M(1) 712 collimates andreflects the light back onto a second detection module D2 and so on andso forth down the serial chain of micro-mirrors and detection modules ofthe transparent block of image array 708. The second detection module715 functions similarly to the first detection module 714.

Reflections progress through the image array 106B,708,708′ in each ofthe first detection module 714 and the second detection module 715 asdescribed herein, alternately focusing and collimating the light withsuccessively shorter band-passes of light through the dichroic filtersinto odd and even detectors 118, respectively in the odd and evendetector channels. Accordingly, different wavelengths are de-multiplexedby the plurality of detectors in each of the first detection module 714and second detection module 715.

For a given fluorescent event, signals from each detector (e.g.,detector 318 shown in FIG. 4B, lens/detector D1 through D16 in FIGS.6-7) is amplified, digitized and synchronized by an electronics systemto provide a spectral representation of the input fluorescent lightsignal. Integration of the detection electronics into the optical moduleassembly allows compact design and lower noise by minimizing thecoupling length of detector and amplification circuit. The detector 318shown in FIG. 4B converts an optical signal, such as the inputfluorescent light signal, into an electrical signal.

FIGS. 7B and 7C respectively illustrate right and left perspective viewsof the dual detection module 700 with the pair of 8-channel compactwavelength detection modules 714,715. Each of the detection modules714,715 includes a mounting base 720 and a cover 722 to enclose amounting block 1200 (see FIG. 12) to which the lens/detectors 711 in thedetector array or chain is mounted. The mounting base 720 and cover 722keeps the elements of the image array 708,708′ in the transparent block806,1006 aligned together with the detector array in each detectormodule 714,715. The mounting base 720 of each detection module 714,715is coupled to the base 710 by a plurality of fasteners.

The input stage 701 includes an optical bench 751 with a plurality offilter slots to receive filters 703-705; a plurality of lens slots toreceive lenses 702,706,713; and one or more light channels along whichlight is reflected and propagates through the filters and lenses. Theoptical bench 751 is coupled to the base 710 of the detection module 700to maintain alignment with the detection modules 714-715.

Referring now to FIG. 13, a top view of an optical plate assembly 1300in a modular flow cytometry system is shown. The optical plate assembly1300 includes a laser system 1370 having three semiconductor lasers1370A,1370B,1370C that direct excitation into a flow cell assembly 1308where a sample fluid flows with sample particles. The laser system 1370attempts to direct the multiple (e.g., three) laser beams in a co-linearmanner toward the flow cell assembly 1308. However, the multiple laserbeams can be slightly offset from one another. The laser system 1370includes semiconductor lasers 1370A,1370B,1370C having wavelengthstypically at about 405 nanometers (nm), 488 nm, and 640 nm. The outputpower of the 405 nm semiconductor laser is usually larger than 30milliwatts (mW); the output power of the 488 nm semiconductor laser isusually greater than 20 mW; and the output power of the 640 nmsemiconductor laser is usually greater than 20 mW. Controllerelectronics control the semiconductor lasers to operate at a constanttemperature and a constant output power.

An optical system spatially manipulates the optical laser beams1371A,1371B,1371C generated by the semiconductor lasers1370A,1370B,1370C respectively. The optical system includes lenses,prisms, and steering mirrors to focus the optical laser beams onto afluidic stream carrying biological cells (bio cells). The focusedoptical laser beam size is typically focused for 50-80 microns (μm)across the flow stream and typically focused for 5-20 μm along thestream flow in the flow cell assembly 1308. In FIG. 13, the opticalsystem includes beam shapers 1330A-1330C that receive the laser light1371A,1371B,1371C from the semiconductor lases 1370A-1370C,respectively. The laser light output from the beam shapers 1330A-1330Care coupled into mirrors 1332A-1332C respectively to direct the laserlight 1399A,1399B,1399C towards and into the flow cell assembly 1308 totarget particles (e.g. biological cells) stained with a dye offlourochromes. The laser light 1399A,1399B,1399C is slightly separatedfrom each other but directly substantially in parallel by the mirrors1332A-1332C into the flow cell assembly 1308.

The laser light beams 1399A,1399B,1399C arrive at the biological cells(particles) in the flow stream in the flow cell assembly 1308. The laserlight beams 1399A,1399B,1399C are then scattered by the cells in theflow stream causing the flourochromes to fluoresce and generatefluorescent light. A forward scatter diode 1314 gathers on-axisscattered light. A collection lens 1313 gathers the off-axis scatteredlight and the fluorescent light and directs them together to adichromatic mirror 1310. The dichromatic mirror 1310 focuses theoff-axis scattering light onto a side scatter diode 1315. Thedichromatic mirror 1310 focuses the fluorescent light onto at least onefiber head 1316. At least one fiber assembly 102 routes the fluorescentlight toward at least one detector module 600,700.

For a more detailed analysis of a biological sample using differentfluorescent dyes and lasers wavelengths, multiple fiber heads 1316,multiple fiber assemblies 102, and multiple detector modules 600,700 canbe used. Three fiber heads 1316A,1316B,1316C can be situated in parallelto receive the fluorescent light and three fiber assemblies102A,102B,102C can be used to direct the fluorescent light to threedetector modules 600A,600B,600C or 700A,700B,700C.

The three fiber heads 1316A,1316B,1316C (and three fiber assemblies102A,102B,102C) are enabled because the three laser light beams599A,599B,599C are slightly offset (e.g., not precisely co-linear).Accordingly, three fiber heads 1316A,1316B,1316C can collect light beamdata separately from the three laser light beams 599A,599B,599C, whichhave three different wavelengths. The three fiber assemblies102A,102B,102C then direct light into three different detector modules(e.g., three different detector modules 600A,600B,600C or700A,700B,700C).

Alternatively, the modular flow cytometry system can use one detectormodule 600,700 to collect the light beam data. For example, the threefiber assemblies 102A,102B,102C can direct light into one detectormodule 600,700, as opposed to three different detector modules.Separation of the light beam data is then handled as data processingoperations, instead of separating the light beam data by using threedifferent detector modules. Using one detector module may be lesscomplex from a physical device standpoint. However, the data processingoperations can be more complex because separation of the light beam datarequires more data manipulation (e.g., identifying different wavelengthsand separating light beam data accordingly).

Cell geometric characteristics can be categorized though analysis of theforward and side scattering data. The cells in the fluidic flow arelabeled by dyes of visible wavelengths ranging from 400 nm to 900 nm.When excited by the lasers, the dyes produce fluorescent lights, whichare collected by a fiber assembly 102 and routed toward a detectormodule 600,700. The modular flow cytometry system maintains a relativelysmall size for the optical plate assembly via compact semiconductorlasers, an 11.5× power collection lens 1313, and the compact image arrayin the detector modules 600,700.

The collection lens 1313 contributes to the design of the detectormodules 600,700. The collection lens 1313 has a short focal length forthe 11.5× power. The collection lens 1313, an objective lens, has a highnumerical aperture (NA) of about 1.3 facing the fluorescence emissionsto capture more photons in the fluorescence emissions over a wide rangeof incident angles. The collection lens 1313 has a low NA of about 0.12facing the collection fiber 102 to launch the fluorescent light into thefiber over a narrow cone angle. Accordingly, the collection lens 1313converts from a high NA on one side to a low NA on the opposite side tosupport the magnification m in the input channel of the detector module600,700.

The diameter of the core of the collection fiber assembly 517 is betweenabout 400 μm and 800 μm, and the fiber NA is about 0.12 for a corediameter of about 600 μm. The fiber output end can be tapered to a corediameter of between about 100 μm and 300 μm for controlling the imagingsize onto the receiving photodiode.

The input end of the collection fiber 102 can also include a lensedfiber end to increase the collection NA for allowing use of a fiber corediameter that is less than about 400 μm. Because the fiber 102 has theflexibility to deliver the light anywhere in the flow cytometer system,the use of fiber for fluorescence light collection enables optimizationof the location of the receiver assembly and electronics for a compactflow cytometer system.

To manufacture a low cost flow cytometer, lower cost components can beintroduced. The image array 106B in each detection module 614,714,715 isformed out of a solid transparent material to provide detection modulethat is reliable, low cost, and compact. Furthermore, the flow cytometeruses low cost off the shelf thin outline (TO) can detectors.

Referring now to FIG. 12, a mounting block 1200 is shown adjacent thetransparent block 806,1006 (see FIGS. 8-11) that are coupled together toform the if image array 708,708′ that is to be mounted by the mountingbase 720 and cover 722 to the base 710 of the compact detector module700 shown in FIGS. 7A-7C. The mounting block 1200 includes a pluralityof angled curved openings 1201 to receive a plurality of TO canlens/detectors 711. The alignment of the mounting block 1200 with thetransparent block 806,1006 of the imaging array 708 and the angle of theangled curved openings 1201 is such that light reflected off amicro-mirror 712E can be bandpass filtered by a dichroic filter 709E andcoupled into a lens/detector 711E.

Each TO can lens/detector 711 of the plurality includes a focusing lens1211 and a low cost TO can detector 1212 coupled together. The TO candetector 1212 includes a window top and a semiconductor photodetector1213 inside the TO can package. The semiconductor photodetector 1213 iselectrically coupled to a plurality of electrical pins 1214 that extendoutside the TO can package to which the other electronics of the flowcytometer electrically couple. Like the detector 318 shown in FIG. 4B,the semiconductor photodetector 1213 converts an optical signal, such asthe input fluorescent light signal, into an electrical signal on atleast one electrical pin 1214.

Referring now to FIG. 8, a perspective view of a transparent block 806formed out of a solid transparent material 800 is shown for anembodiment of the if image array 106B,608,708,708′. The solidtransparent material 800 used for the transparent block 806 can be clearglass or clear plastic, for example. A plurality of micro-mirrors 810 ina row and serial chain is formed into or on one side of the transparentblock 806 of transparent material 800. A plurality of dichroic orbandpass filters 812 in a row and serial chain are formed into or on anopposing side of the transparent block 806 of transparent material 800.Each dichroic or bandpass filter 812 is tuned to a different range ofwavelengths of light to allow detection of a broad range of fluorescentlight being emitted by flourochromes. In one embodiment, the pluralityof micro-mirrors 810 are concave spherical mirrors.

The transparent block 806 formed out of the solid transparent material800 further includes a 12-degree wedge face 820 to receive light from afocusing lens, such as described with the if imaging array 708. Thelight enters normal to the surface of the wedge face 820 and is directed(bent) towards the first dichroic or bandpass filter D(1). The lightpasses through the transparent block 806 to reach the first dichroic orbandpass filter D(1).

Referring now to FIG. 10, a perspective view of a transparent block 1006is shown formed out of the solid transparent material 800 for anotherembodiment of the if image array 106B,608,708,708′. The solidtransparent material 800 used for the transparent block 1006 can beclear glass or clear plastic, for example. A plurality of micro-mirrors1010 are concave rectangular mirrors formed into a side of thetransparent material. A plurality of dichroic or bandpass filters 1012are formed into or on an opposing side of the transparent material 800.Each dichroic or bandpass filter 812 is tuned to a different range ofwavelengths of light to allow detection of a broad range of fluorescentlight being emitted by flourochromes.

The solid transparent material 800 further includes a 12-degree wedgeface 820 to receive light from a focusing lens, such as described withthe image array 708. The light enters normal to the surface of the wedgeface 820 and directed (bent) towards the first dichroic or bandpassfilter D(1). The light passes through the block to reach the firstdichroic or bandpass filter D(1).

Referring now to FIG. 9A, a cross section view illustrates the distance(e.g., thickness L) between the spherical micro-mirror 810 on one sideand the opposite side of the transparent block 806. An axis 814perpendicular to the transparent block at the center of the sphericalmicro-mirror 810 extends out to the opposite side of the transparentblock 806. FIG. 9B illustrates an axis 815 perpendicular to thetransparent block at the center of the dichroic or bandpass filter 812.The axis 815 extends out to the opposite side of the transparent block806 of transparent material 800. The axes 814 and 815 are parallel toeach other.

A reflective material 811 is formed (e.g., disposed) on a sphericaltransparent micro-lens shape of the solid transparent material 800 toform each spherical micro-mirror 810 in one side of the transparentblock 806. The dichroic or bandpass filter 812 is coupled to thematerial 800 in an opposite side of the transparent block 806.

FIG. 11 similarly shows the distance and the axis 1014 between theconcave rectangular micro-mirror 1010 on one side and the opposite sideof the transparent material 800 forming the transparent block 1006. FIG.11 further illustrates an axis 1015 perpendicular at a center point ofthe dichroic or bandpass filter 1012. The optical axis 1015 extends outto the opposite side of the transparent block 1006 formed by thetransparent material 800. The optical axes 1014 and 1015 are parallel toeach other.

FIG. 11 further shows a reflective material 1011 is formed (e.g.,disposed) on a curved transparent rectangular shape of the solidtransparent block 1006 formed out of the transparent material 800 toform the rectangular micro-mirror 1010. The dichroic or bandpass filter1012 is coupled to an opposite side of the solid transparent block 1006.

The fluorescence dyes used in flow cytometry applications covers theentire visible and near infrared wavelength range. The emissionwavelength bandwidth is typically large for long wavelengthfluorochromes. Each of the dichroic or bandpass filters 812 can havetheir detector filter passbands and center wavelength optimized tomeasure different dyes with the same amount of spectral sampling.Furthermore, individual filter optimization allows exclusion ofexcitation wavelengths from other lasers. In this way, the detector ineach channel can be utilized fully to detect the signal of interest. Inconjunction with a fluorescence spectral unmixing algorithm executed bya processor of a computer, the individual and optimized passbanddetection provides the ultimate detection of a large number offluorescent dyes of interest.

METHODS

Methods of using the various detection systems disclosed herein in aflow cytometer are now described. Before launching the fluorescent lightgenerated by flourochromes excited by laser light out of the end of theoptical fiber 102 shown in the figures, the fluorescent light ofdifferent wavelengths is generated by various flourochromes markingdifferent particles in a sample in a flow channel that are excited bylaser light. The fluorescent light that is generated is received by acollection lens near the opposite end of the laser as can be seen inFIG. 13. A converter is used to convert from a first numeric aperture onthe capture side to a second numeric aperture less than the firstnumeric aperture to better match the numeric aperture of the opticalfiber. The optical fiber then directs the fluorescent light towards theend of the optical fiber to flexibly direct it towards the compactdetection module 600,700.

The optical fiber 102 couples the fluorescent light into the end of theoptical fiber thereby launching it out from the optical fiber. Thelaunched fluorescent light has different wavelengths generated by thedifferent flourochromes attached to different particles in the samplefluid that have been excited by laser light.

In an input channel, the light launched from the end of the opticalfiber is collimated and focused by a lens towards the first dichroicfilter of a first plurality of dichroic filters in a firstde-multiplexing imaging array.

Further along the input channel, the laser light used to excite thedifferent flourochromes that is launched from the optical fiber isblocked from interfering with detecting the wavelengths of fluorescentlight by a blocking device.

Further along the input channel, an image size from an end of theoptical fiber is magnified to a spot size for a first dichroic filter ina serial chain or row of the first plurality of dichroic filters in thefirst de-multiplexing imaging array.

In the first de-multiplexing imaging array, a first wavelength range ofthe fluorescent light is alternatively reflected between the firstplurality of dichroic filters and a serial chain or row of a firstplurality of micro-mirrors to collimate the fluorescent light on oddnumbered dichroic filters and re-image the fluorescent light on evennumbered dichroic filters. A focal length of the first plurality ofmicro-mirrors and a distance of separation between the first pluralityof dichroic filters and the first plurality of micro-mirrors provides atelescopic effect along the chain of micro-mirrors to collimate thefluorescent light on the odd numbered dichroic filters and re-image thefluorescent light on the even numbered dichroic filters.

In the serial chain or row of the first plurality of dichroic filters,different wavelength ranges of the first wavelength range of thefluorescent light is band passed at each to de-multiplex the wavelengthspectrum of the first wavelength range of the fluorescent light.

Adjacent the serial chain or row of the first plurality of dichroicfilters is a serial chain or row of plurality of detector channels asshown in FIGS. 3, 6, and 7A-7C with a first plurality of firstdetectors. Each detector channel has a lens to focus the differentwavelength ranges of the fluorescent light into a first plurality oflight detectors.

The serial chain or row of plurality of detectors detects thefluorescent light in each of the different wavelength ranges of thefirst wavelength range associated with each flourochrome tagged to aparticle. The plurality of light detectors convert the fluorescent lightreceived by each into an electrical signal that can be analyzed andcounted.

With the fluorescent light converted into an electrical signal by thedetectors, a computer with a processor can then be used to count anumber of each of the different particles in the sample fluid such asdisclosed in application Ser. No. 15/498,397 titled COMPACT MULTI-COLORFLOW CYTOMETER filed on Apr. 26, 2017 by David Vrane et al.,incorporated herein by reference.

A second and/or third de-multiplexing imaging array may be used inparallel with the first de-multiplexing imaging array. In this case, themethods further include splitting the fluorescent light into the firstwavelength range of the fluorescent light for the first de-multiplexingimaging array, a second wavelength range of the fluorescent light forthe second de-multiplexing imaging array, and/or a third wavelengthrange of the fluorescent light for the third de-multiplexing imagingarray. Such as shown in FIG. 13, a first optical fiber 102A may be usedto direct fluorescent light towards the first de-multiplexing imagingarray. A second optical fiber 102B may be used to direct fluorescentlight towards the second de-multiplexing imaging array. A third opticalfiber 102B may be used to direct fluorescent light towards the thirdde-multiplexing imaging array.

The steps described herein for the first de-multiplexing imaging arraymay be concurrently performed by the second and/or third de-multiplexingimaging arrays so different additional ranges of wavelengths may beanalyzed. For brevity, the repeated steps are not repeated butincorporated here by reference.

CONCLUSION

The embodiments are thus described. While embodiments have beenparticularly described, they should not be construed as limited by suchembodiments, but rather construed according to the claims that followbelow.

While certain exemplary embodiments have been described and shown in theaccompanying drawings, it is to be understood that such embodiments aremerely illustrative of and not restrictive on the broad invention, andthat the embodiments not be limited to the specific constructions andarrangements shown and described, since various other modifications mayoccur to those ordinarily skilled in the art.

Certain functions of a flow cytometer can be implemented in software andexecuted by a computer or processor, such as the analysis of theelectrical signals detected by the detectors to count differentparticles in a sample fluid. The program or code segments of thesoftware are used to perform the necessary tasks to perform thosefunctions. The program or code segments can be stored in a processorreadable medium or transmitted by a computer data signal embodied in acarrier wave over a transmission medium or communication link. Theprocessor readable medium can include any storage medium that can storeinformation. Examples of the processor readable medium include anelectronic circuit, a semiconductor memory device, a read only memory(ROM), a flash memory, an erasable programmable read only memory(EPROM), a floppy diskette, a CD-ROM, an optical disk, and a hard disk.The code segments can be downloaded via computer networks such as theInternet, Intranet, etc. to the storage medium.

While this specification includes many specifics, these should not beconstrued as limitations on the scope of the disclosure or of what maybe claimed, but rather as descriptions of features specific toparticular implementations of the disclosure. Certain features that aredescribed in this specification in the context of separateimplementations can also be implemented in combination in a singleimplementation. Conversely, various features that are described in thecontext of a single implementation can also be implemented in multipleimplementations, separately or in sub-combination. Moreover, althoughfeatures can be described above as acting in certain combinations andeven initially claimed as such, one or more features from a claimedcombination can in some cases be excised from the combination, and theclaimed combination can be directed to a sub-combination or variationsof a sub-combination. Accordingly, the claimed invention is limited onlyby patented claims that follow below.

1. A flow cytometer comprising: a compact light detection moduleincluding a first image array having a transparent block, a plurality ofmicro-mirrors in a row coupled to a first side of the transparent block,and a plurality of filters in a row coupled to a second side of thetransparent block opposite the first side, wherein each of the pluralityof filters reflects light to one of the plurality of micro-mirrors andpasses light of a differing wavelength range and each of the pluralityof micro-mirrors reflects light to one of the plurality of filters, suchthat incident light into the image array zigzags back and forth betweenconsecutive filters of the plurality of filters and consecutivemicro-mirrors of the plurality of micro-mirrors, wherein a radius ofcurvature of each of the plurality of micro-mirrors directs light ontoeven filters and reimages light onto odd filters of the plurality offilters along the row.
 2. The flow cytometer of claim 1, wherein thecompact light detection module further includes a first plurality ofdetector channels in a row forming a first detector chain adjacent thefirst image array, the first plurality of detector channels respectivelyin light communication with the plurality of filters of the first imagearray, wherein each detector channel includes a focusing lens, and aphoto detector.
 3. The flow cytometer of claim 2, wherein the compactlight detection module further includes an input channel that imagesfrom an aperture at a fiber to a surface of a first filter of the firstplurality of filters; and the flow cytometer further comprises acollection lens with a first side facing a sample chamber and anopposing second side, the first side of the collection lens having afirst numeric aperture (NA) and the second side of the collection lenshaving a second NA less than the first NA; and an optical fiber with afirst end aligned with an optical axis of the collection lens and asecond end aligned with an optical axis of the input channel of thecompact detection module, the optical fiber having a fiber NA greaterthan or equal to the second NA of the collection lens.
 4. The flowcytometer of claim 3, wherein the input channel magnifies the size ofthe input image size from the aperture to the surface of the firstfilter.
 5. The flow cytometer of claim 3, wherein the input channelincludes a collimating lens to receive launched fluorescent light andcollimate the light, a blocking filter to reject a laser light used tostimulate fluorochromes in the collimated light, and a focusing lens tofocus the collimated light from the collimating lens onto the firstfilter.
 6. The flow cytometer of claim 1, wherein the compact lightdetection module further includes a second image array having atransparent block, a plurality of micro-mirrors in a row coupled to afirst side of the transparent block, and a plurality of filters in a rowcoupled to a second side of the transparent block opposite the firstside.
 7. The flow cytometer of claim 6, wherein the compact lightdetection module further includes a second plurality of detectorchannels in a row forming a second detector chain adjacent the secondimage array, the second plurality of detector channels respectively inlight communication with the plurality of filters of the second imagearray, wherein each detector channel includes a focusing lens, and aphoto detector.
 8. The flow cytometer of claim 7, wherein the compactlight detection module further includes an input channel that couples aninput image from an aperture at a fiber to a surface of a first filterof the first plurality of filters and a surface of a first filter of thesecond plurality of filters; and the flow cytometer further comprises acollection lens with a first side facing a sample chamber and anopposing second side, the first side of the collection lens having afirst numeric aperture (NA) and the second side of the collection lenshaving a second NA less than the first NA; and an optical fiber with afirst end aligned with an optical axis of the collection lens and asecond end aligned with an optical axis of the input channel of thecompact detection module, the optical fiber having a fiber NA greaterthan or equal to the second NA of the collection lens.
 9. The flowcytometer of claim 8, wherein the input channel magnifies the size ofthe input image size from the aperture to the surface of the firstfilter of each of the first and second plurality of filters.
 10. Theflow cytometer of claim 8, wherein the input channel includes acollimating lens to receive launched fluorescent light and collimate thelight, a blocking filter to reject a laser light used to stimulatefluorochromes in the collimated light, a beam splitter to split thecollimated light into a first range of wavelengths and a second range ofwavelengths different from the first range, a first focusing lens tofocus the collimated light with the first range of wavelengths onto thefirst filter of the first plurality of filters, and a second focusinglens to focus the collimated light with the second range of wavelengthsonto the first filter of the second plurality of filters.
 11. The flowcytometer of claim 1, wherein a radius of curvature of the plurality ofmicro-mirrors forms a focal length equal to a beam path length frommicro-mirror to opposing filter.
 12. The flow cytometer of claim 1,wherein each of the plurality of filters is a dichroic filter.
 13. Theflow cytometer of claim 1, wherein each of the plurality of filters is abandpass filter.
 14. The flow cytometer of claim 1, wherein each of theplurality of filters are a combination of a dichroic filter and abandpass filter coupled in light communication.
 15. The flow cytometerof claim 1, wherein wherein the light directed by each of the pluralityof micro-mirrors is collimated onto even filters.
 16. An apparatuscomprising: a first light detection module to demultiplex a firstfluorescent light in a first range of wavelengths, the first lightdetection module including a first plurality of detectors in a firstrow; a first transparent block adjacent the first plurality ofdetectors, a first plurality of micro-mirrors coupled to a first side ofthe first transparent block, and a first plurality of filters in a firstrow coupled to a second side of the first transparent block opposite thefirst side, each of the first plurality of filters to reflect light toone of the first plurality of micro-mirrors and pass light of adifferent wavelength range towards one of the first plurality ofdetectors.
 17. The apparatus of claim 16, further comprising: a secondlight detection module to demultiplex a second fluorescent light in asecond range of wavelengths, the second light detection module includinga second plurality of detectors in a second row; a second transparentblock adjacent the second plurality of detectors, a second plurality ofmicro-mirrors coupled to a first side of the second transparent block,and a second plurality of filters in a second row coupled to a secondside of the second transparent block opposite the first side, each ofthe second plurality of filters to reflect light to one of the secondplurality of micro-mirrors and pass light of a different wavelengthrange towards one of the second plurality of detectors.
 18. Theapparatus of claim 16, further comprising: an input channel coupled inlight communication with the first light detection module and the secondlight detection module, the input channel to receive a fluorescent lightbeam and split it into the first fluorescent light in the first range ofwavelengths and the second fluorescent light in the second range ofwavelengths.
 19. The apparatus of claim 18, wherein the input channelincludes a wavelength beam splitter (other than power beam splitter) tosplit the fluorescent light beam into the first fluorescent light andthe second fluorescent light.
 20. The apparatus of claim 18, wherein theinput channel further includes a collimating lens to receive thefluorescent light from an optical fiber and collimate the fluorescentlight, and a blocking filter to reject a laser light used to stimulatefluorochromes in the fluorescent light and pass the fluorescent light tothe beam splitter.
 21. The apparatus of claim 20, wherein the inputchannel further includes a first focusing lens in light communicationwith the beam splitter, the first focusing lens to focus the firstfluorescent light onto a first filter of the first plurality of filters,and a second focusing lens in light communication with the beamsplitter, the second focusing lens to focus the collimated light withthe second range of wavelengths onto a first filter of the secondplurality of filters. 22-29. (canceled)